A major goal of tissue engineering is to provide replacements for malformed, damaged, diseased or worn-out parts of the body. This requires either the accurate reconstitution of body parts, either in vitro or in vivo; or the construction of surrogate body parts of another design which, however, meets the same physiological or structural needs. Efforts at organ reconstitution will be most effective if they are based upon an understanding of the principles which determine the organization of cells into properly structured tissues and organs. Some thirty years ago, this laboratory laid the theoretical foundations for such an understanding with the proposition that tissue surface/interfacial tensions, reflecting the relative adhesive intensities at cell interfaces, determine important elements of organ design: whether two cell populations preferentially intermix or segregate; which will tend to envelop the other and to what extent. These principles were codified in the "differential adhesion hypothesis" (DAH), for which considerable experimental evidence was adduced. We have recently utilized genetic engineering techniques to introduce into originally non-adhesive cells cDNAs coding for the synthesis and regulated expression of specific adhesion molecules, thus generating new cell lines with predetermined adhesive properties. When two such specially designed cell lines were cultured together, they re-assembled to form organ-like structures with one of the cell lines forming the "medulla" and the other the "cortex": the precise structure predicted in advance by the DAH. We propose now to generate a series of such genetically engineered cell lines designed to test a series of specific predictions of the DAH. Our ultimate goals are to solidify the principles specifying the assembly of cells into tissue constructs of specific design and to demonstrate the ability of genetic engineering techniques to program cells to assemble in deliberately chosen anatomical positions. (1) We will engineer cell lines displaying defined amounts of known adhesion molecules. (2) We will measure the densities (sites/cell) of these adhesion molecules. (3) We will utilize these cell lines to test specific predictions of the DAH concerning their mutual assembly behavior when combined. (4) We will measure the surface tensions (reflecting aggregate cohesiveness) of the engineered cell aggregates, these being the theoretical determinants of cell sorting and tissue spreading behaviors, and correlate these with the measured adhesive site densities. (5) We will investigate whether the intercellular adhesive interactions theoretically underlying these cell aggregate surface tensions, including interactions between two cell monolayers expressing different amounts of various adhesion molecules, can be quantified directly by distractive measurements. We anticipate application of both the principals and the techniques employed here to the design of "man-made" organs engineered to carry out specific functions in the human body.